Rapid Screening Assay for Qualitative Detection of Multiple Febrile Illnesses

ABSTRACT

A single-use multiplex, or assay, screening test for the detection of one or more of a plurality of unrelated febrile illnesses is provided. The febrile illnesses for which the test is designed are unrelated, in that the illnesses may be caused, by way of example, by infection from viruses, bacterium and/or parasites; by infection from viruses, bacterium, parasites or other contagions that are animal borne; by infection from viruses, bacterium, parasites, or other contagions that can be aerosolized for transmission; by infection from viruses, bacterium, parasites or other contagions that are transmitted from direct contact; by infection from viruses, bacterium, parasites or other contagions that are generally transmitted in the tropics and/or subtropics; and/or by infection from a virus, bacteria, parasite sharing one or more related feature and which causes a febrile illness. The assay test provides rapid results to a point of care center or other facility requiring such results to facilitate treatment and or containment of the illnesses.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to immunochromatographic, rapid screeningtest for the in vitro detection of illnesses from a bodily fluid,including, but not limited to, blood. More particularly, the test is aqualitative assay test for the quick screening of multiple febrileillnesses from the bodily fluid.

2. State of the Art

A sudden and often severe fever is indicative of a febrile illness. Inmany parts of the world, febrile illnesses are often misdiagnosed due tothe inherent variability associated with febrile-related diseases.Misdiagnosis is followed by presumptive treatment which may not addressthe causative infection. Improper treatment and control may allowinfectious febrile illnesses to spread through populations. Febrileillnesses with potentially high morbidity and mortality include but arenot limited to Ebola, Malaria, Dengue fever, Plague, and Melioidosis.

Ebola, previously known as Ebola hemorrhagic fever, is a rare and deadlydisease caused by infection with one of the Ebola virus strains. Ebolacan cause disease in humans and nonhuman primates (monkeys, gorillas,and chimpanzees). Ebola is caused by infection with a virus of thefamily Filoviridae, genus Ebolavirus. There are four identified Ebolavirus species known to cause disease in humans: Ebola virus (Zaireebolavirus); Sudan virus (Sudan ebolavirus); Tai Forest virus (TaiForest ebolavirus, formerly Cote d'Ivoire ebolavirus); and Bundibugyovirus (Bundibugyo ebolavirus). Researchers believe that the virus isanimal-borne and that bats are the most likely reservoir. Transmissionoccurs through direct contact with blood or other body fluids, objectscontaminated with the virus and infected animals. Early symptoms such asfever are non-specific and often seen in patients with other illnesses,including, e.g., Malaria.

Malaria results from infection with the parasite Plasmodium. ThePlasmodium parasite is transmitted via the bite of an infected Anophelesmosquito. The infected mosquito deposits parasites onto its human host,which then invade the host's liver followed by the red blood cells. Therupturing of infected red blood cells release parasites into the bloodstream, giving rise to Malaria symptoms such as nausea, fever, vomiting,headache, sweating, and chills. These flu-like symptoms can be mild,severe, or even fatal.

The Dengue virus is a single-stranded RNA virus from the Flaviviridaefamily that can cause Dengue Fever, Dengue hemorrhagic fever, and/orDengue shock syndrome. Dengue is considered to be one of the leadingcauses of illness in tropical and subtropical regions. It is estimatedthat nearly 100 million people are infected with Dengue on an annualbasis. Dengue fever, the most common presentation of infection withDengue virus, is caused by any of the four Dengue serotypes (Dengue 1,2, 3,; or 4). Transmission is carried out by the mosquito vectors Aedesaegypti and Aedes albopictus.

Infection with the gram-negative bacterium Burkholderia pseudomalleioften presents as the tropical disease Melioidosis, also known asWhitmore's disease, which is found primarily in Southeast Asia andAustralia. The bacteria are spread through direct contact with orinhalation of contaminated water or soil. Localized, pulmonary,bloodstream, or disseminated infection can occur and symptoms, such asfever, pain, ulceration, cough, respiratory distress, weight loss,headache, or seizures, generally appear two to four weeks afterexposure. However, there are indications that the bacteria may remainlatent in a host for up to several years. The infection, characterizedmost often by the CPS antigen, can be treated with appropriateantimicrobial therapy when diagnosed properly. Due to the severity ofillness and its aerosol transmission, there is concern regarding the useof Burkholderia pseudomallei as a bioterrorism agent.

Yersinia pestis is a gram negative, rod-shaped bacterium that results inPlague. It is found in rodents and their fleas, and occurs in many partsof the world including the United States. Historically, three largepandemics have killed approximately 200 million people. While largeoutbreaks are now rare, a few cases of plague still arise in endemicareas around the world including the southwestern region of the UnitedStates. There are three forms of plague: pneumonic, bubonic andsepticemic plague. Bubonic plague is the most common, while pneumonicplague is considered to be the most likely to be encountered in abioterrorism event. The pneumonic form of the disease occurs whenYersinia pestis infects the lungs. It can be transmitted through the airwhen a person breathes in aerosolized bacteria. With pneumonic plague,the first signs of illness are fever, headache, weakness and a rapidlydeveloping pneumonia. The pneumonia progresses for 2 to 4 days and maycause respiratory failure and shock. To reduce the likelihood of death,antibiotics must be administered within the first 24 hours; thus, earlydiagnosis is essential.

All of the above illnesses are non-specifically characterized by a highgrade fever.

Many types of ligand-receptor assays have been used to detect thepresence of various substances that are indicative of a bodily responseto an illness state. Such substances, often generally called ligands,are present in body fluids such as blood, urine, or saliva. These assaysinvolve antigen antibody reactions, synthetic conjugates comprisingradioactive, enzymatic, fluorescent, or visually observable polystyreneor metal sol tags, and specially designed chambers in which the antigensand antibodies react. In all these assays, there is a receptor, e.g., anantibody, which is specific for the selected ligand or antigen, and ameans for detecting the presence, and in some cases the amount, of theligand-receptor reaction product. Some tests are designed to make aquantitative determination, but in many circumstances all that isrequired is a positive/negative qualitative indication. A visuallyobservable indicator such as the presence of agglutination or a colorchange is preferred.

Even the qualitative assays must be very sensitive because of the oftensmall concentration of the ligand of interest in the test fluid. Falsepositives can also be troublesome, particularly with agglutination andother rapid detection methods such as dipstick and color change tests.Because of these problems, so-called “sandwich” assays and othersensitive detection mechanisms which use metal sols or other types ofcolored particles have been developed.

In a “sandwich” assay, a target analyte such as an antigen is“sandwiched” between a labeled antibody and an antibody immobilized ontoa solid support. The assay is read by observing the presence and/oramount of bound antigen-labeled antibody complex. In a “competition”immunoassay, antibody bound to a solid surface is contacted with asample containing an unknown quantity of antigen analyte and withlabeled antigen of the same type. The amount of labeled antigen bound onthe solid surface is then determined to provide an indirect measure ofthe amount of antigen analyte in the sample.

Because these and other assays can detect both antibodies and antigens,they are generally referred to as immunochemical ligand-receptor assaysor simply immunoassays.

Solid phase immunoassay devices, whether of the sandwich or competitiontype, provide sensitive detection of an analyte in a biological fluidsample such as blood, urine, or saliva. Solid phase immunoassay devicesincorporate a solid support to which one member of a ligand-receptorpair, usually an antibody, antigen, or hapten, is bound. Common earlyforms of solid supports were plates, tubes, or beads of polystyrenewhich were well known from the fields of radioimmunoas say and enzymeimmunoassay. More recently, a number of porous materials such as nylon,nitrocellulose, cellulose acetate, glass fibers, and other porouspolymers have been employed as solid supports.

A number of self-contained immunoassay kits using porous materials assolid phase carriers of immunochemical components such as antigens,haptens, or antibodies have been described. These kits are usuallydipstick, flow-through, or migratory in design. One test kit isdisclosed in co-owned U.S. Pat. No. 7,879,597 to Esfandiari which ishereby incorporated by reference herein in its entirety. This referenceteaches an accurate self-contained immunoassay that can be used byminimally trained personnel to obtain valid qualitative assay results.The test also requires a relatively small amount of ligand molecule andcan be manufactured at low cost. In addition, the test can be operatedin a sensitive manner with a small sample volume while providingaccurate results.

SUMMARY

Early and appropriate diagnosis of febrile illnesses and infection mayallow for effective treatment options against associated complicationsand may also prevent transmission of the diseases.

A single-use multiplex, or assay, screening test for the detection offebrile illnesses is provided. The febrile illness assay test isintended for use as a point-of-care device to aid in the diagnosis ofseveral severe febrile illnesses using a body fluid such a fingerstickwhole blood. The assay test provides relatively quick results andfacilitates clinical decision making.

According to one aspect, the early manifestation of many febrilediseases is very similar. Accordingly, a multiplex assay that candistinguish among febrile diseases at an early stage can enable a moreefficient rapid response.

The assay test is an in vitro qualitative detection test preferablyutilized for the presumptive detection of infection with one or more ofvarious febrile illnesses, i.e., to detect (“positive”) or reject(“negative”) the illnesses.

In one embodiment, the assay test is adapted to provide presumptive(i.e., confirmatory) detection of at least two unrelated febrileillnesses. In one embodiment, the assay test is adapted to providepresumptive detection of at least three unrelated febrile illnesses. Thefebrile illnesses can be selected for testing on the assay using commonfeatures of (or alternatively dissimilar features relative to) the twoor more of the unrelated febrile illnesses. By way of example, theselection of febrile illnesses for which the test is designed mayinclude illnesses caused by one or more of the following: viruses,bacterium and/or parasites (different pathogens); viruses, bacterium,parasites or other contagions that are animal borne, whether selected tobe animal borne by the same animal (mosquitos) or by different animals(e.g., bat, rodent, mosquito, flea); viruses, bacterium, parasites, orother contagions that can be aerosolized for transmission; viruses,bacterium, parasites or other contagions that are transmitted fromdirect contact; viruses, bacterium, parasites or other contagions thatare generally transmitted in the tropics and/or subtropics; virus,bacteria, parasite sharing one or more related features and which causesa febrile illness.

It is specifically recognized that the febrile illnesses tested by theassay test do not share any particularly common cause for theirrespective disease states. That is, the positive detection of onefebrile illness is not presumptive, indicative or otherwise suggestiveof testing positive for a second febrile illness which is the subject ofthe assay test. Febrile illnesses under the test are epidemiologicallyindependent; i.e., are known to have different etiologies. That is,there is no known pattern, causation, or other relationship from oneillness to the other because each results from at least a differentpathogen. As such, to the extent the test provides a positive detectionfor one febrile illness, it is expected that only one positive detectionfor a febrile illness will be indicated on any one test. It is, ofcourse, possible that a test sample can provide positive detection oftwo or even more febrile illnesses. But given the lack of relationshipof the febrile illnesses for which the test is administered, suchresults would not be expected. Nonetheless, it is anticipated that theunrelated febrile illnesses for which any test is designed will have arationale in their selection for inclusion together on a test.

In accord with one aspect of the assay test, the febrile illnessesdetectable by the assay test may be linked by those febrile illnesseshaving a prevalence within a geographical proximity (by of example,within a state, or within a country, or within neighboring countries, orwithin proximate countries, or within a continent, or within 20°latitude of the equator, or as bounded by geological formationsincluding rivers and/or mountains and/or valleys, or subject toenvironmental disaster, or geopolitical hardships, or othergeographically definable boundary), and/or those febrile illnesseshaving a prevalence within a population in a given time period, and/orthose febrile illnesses to which a group or population may be subject(by way of example, general population of western Africa, or aid workersof one or more relief organizations, whether localized or scattered, orinhabitants of one or more temporary or semi-permanent shelter orhousing systems, including shelter or housing established by the UnitedNations or another relief aid organization), and/or those febrileillnesses which are potentially borne by travelers at a point of entryat a geographical location (e.g., air travelers at an airport arrivingfrom selected points of origination or going to selected points ofdestination, or similar sea travelers at a sea port). For each of thepotential groups identified above, an assay test may be designed to testfor febrile illnesses that are appropriate for the circumstance.

By way of example, the febrile illnesses detected can include EbolaVirus (viral infection and animal borne), Plasmodium parasites(Malaria)(parasitic infection and animal borne by mosquito), Denguevirus (viral infection, animal borne by mosquito, andtropical/subtropical), Yersinia pestis (Plague)(bacterial infection,animal borne by rodent and their fleas, specific endemic regions, can bein aerosol form), Burkholderia pseudomallei (Melioidosis)(bacterialinfection, tropical illness, can be in aerosol form or in contaminatedsoil). Other febrile illnesses that may be tested for include, by way ofexample, Anthrax, Lassa fever, Marburg hemorrhagic Fever, Leptospirosis,Rickettsial disease, Tularemia, Thyphoid, Chikungunya, Coxiella burnetiibacteria (Q-fever), Meningococcal, Pneomococcus, and Crimean-Congohaemorrhagic fever (CCHF).

In accord with embodiments of the assay, and not by way of limitation,the assay is adapted to detect for at least three of, or all of, EbolaVirus, Plasmodium parasites (Malaria), Dengue virus, as well as thebacteria Yersinia pestis (Plague) and Burkholderia pseudomallei(Melioidosis) in a bodily fluid. In accord with one embodiment of theassay test, the assay is adapted to use the bodily fluid of fingerstickwhole blood, venous whole blood, serum and/or plasma. In one aspect,these febrile illnesses are chosen to be part of a single assay becauseof their potential use in bioterrorism.

In accord with an embodiment, the febrile illness assay test includes atleast one lateral flow sorbent material test strip having a first widthand a first length substantially longer than said first width, anddefining a lateral flow pathway along its length. A test site isprovided on or in the test strip with distinct test lines, eachcomprising a ligand-binding protein or particle adapted to couple withan antigen (antibody) of a distinct one of the febrile illnesses undertest. Spaced apart from the test site are a plurality of conjugatesadapted to combine with an antigen (or antibody) of the respectivefebrile illnesses under test. The conjugates include colloidal gold dyeparticles, and are bound to the test strip in a solid phase. Ligandbinding proteins and their conjugates for use in a test cell aredescribed in U.S. Pat. No. 5,714,389 to Charlton, the teaching of whichis hereby incorporated by reference herein in its entirety. The teststrip is provided in a housing, and includes a clear test window over atesting area under which results of the tests are displayed. Anoptionally diluted sample (sample and buffer) is applied to the teststrip and allowed to flow across the test lines. The antibodies (orantigens), if any, in the sample laterally flow to the test site andcombine with the antigens (or antibodies) immobilized at the respectivetest lines. The conjugates are also released from the test strip andcaused to laterally flow to the test site. The conjugates are capturedby antibodies (or antigens) of the febrile illnesses which are retainedat the respective test lines. In a positive test, a conjugated antigen(or antibody) migrates along the test strip and is captured by itsrespective antigen-bound antibody at one of the test lines, producing avisible, colored line to positively indicate a febrile illness. The testlines are keyed to indicate which febrile illness is associated witheach test line. It is appreciated that the test may be indicative of oneor more febrile illnesses, on the showing of one or more colored linesat the test site. In the absence of the antibodies (or antigens)indicative of the febrile illnesses of interest, no associated coloredline would be observed under the test window area.

In accord with one embodiment of the assay test, the assay test includesa first sorbent material having a first location for receiving a buffersolution (in the case of a dry conjugate system) or a conjugate solution(in the case of a liquid conjugate system) with the first sorbentmaterial defining a first horizontal flow path, a second sorbentmaterial having a second location for receiving a sample with the secondsorbent material defining a second horizontal flow path distinct fromthe first flow path, and a plurality of longitudinally displaced testlines or test sites with immobilized antigens or antibodies or otherligand binding molecules such as aptamers, nucleic acids, etc.associated with a plurality of distinct febrile illnesses, all locatedin a test zone at a junction of the first and second sorbent materials.For purposes herein, the term “distinct” when used in conjunction withthe words “flow path” or “migration path” shall be understood to mean“not in fluid communication except via a test zone”. Where the test cellof the invention is provided in a housing, the housing is provided witha first opening adjacent the first location and a second openingadjacent the second location. A viewing window is provided in thehousing above the test zone. In an embodiment, the first sorbentmaterial and second sorbent material are separate pieces which overlieone another and the test line is printed on one or both of the sorbentmaterials at the junction. Alternatively, although not preferred, thefirst and second sorbent materials can be integral with each other. Thesystems of the invention preferably also include a control line or sitewhich may be seen from the viewing window. According to an embodiment,the sorbent materials are laid out in a T shape, where the firstlocation for receiving the buffer or buffer-conjugate solution islocated near one end of the top bar of the T, the second location forreceiving the sample is located near the end of the stem of the T, andthe sorbent materials overlie each other at the intersection. Of course,the sorbent materials may be laid out in other configurations. Thematerials, thicknesses and lengths of the first and second sorbentmaterials are chosen to adjust the timing regarding the liquid sampleand liquid buffer reaching the test site. In the dry conjugate system ofthe invention, a dry conjugate is provided between the first opening andthe test site. The conjugate is supported on or within the sorbentmaterial such that when a buffer is added in the first opening, thesorbent material wicks the buffer to the conjugate which is then carriedby the buffer to the test site. In the liquid conjugate system of theinvention, a buffer-conjugate liquid subsystem is provided and appliedto the first opening. The sorbent material then wicks thebuffer-conjugate subsystem to the test site.

According to a method of using the test cell, sample of interest isprovided to the second opening or location and allowed to travel to thetest site. If any antigens or antibodies are present in the sample, theantigens or antibodies will bind with respective ligand-bindingmolecules at an associated test line. After a desired amount of time toensure the sample has traveled to the test site, a liquid such as abuffer solution is added to the first opening or location. If thesorbent material is supporting a conjugate (i.e., in a dry conjugatesystem), the liquid is preferably simply a buffer solution. If thesorbent material is not supporting a conjugate (i.e., in a liquidconjugate system), the liquid is preferably a buffer-conjugate liquidsubsystem. In any event, after sufficient time to permit the conjugateto migrate to the test site (and control site if provided), the testsite (and control site if provided) is inspected in order to determinewhether the sample is “positive” or not. In a “positive” sample,conjugate have bound with each antigen or antibody attached at the testlines and associated with a respective one of the febrile illnesses. Ina positive sample, at least one test line corresponding to at leastfebrile illness and the control line will be colored. In a negativesample, none of the test lines corresponding to a febrile illness iscolored, but the control line is colored.

In accord with another embodiment of the assay test, the assay testincludes three sorbent test materials, one to initially receive thediluted sample, and one each for the detection of different stageantibodies for a plurality of febrile illnesses. This expeditesdetection of one or more of the plurality of febrile illnesses atdifferent stages of the illness, for example, within a few days aftersymptoms begin and later in a disease course or after recovery.

The assay test includes a test cell having a first buffer-receivinglocation which receives a buffer solution and a first sorbent materialdefining a first horizontal flow path for the first buffer solution, asecond sorbent material defining a second horizontal flow path distinctfrom the first horizontal flow path for the same or a different buffersolution provided to the first buffer-receiving location or to a secondbuffer-receiving location, a third sorbent material defining a thirdhorizontal flow path for a sample provided at a sample-receivinglocation, the third horizontal flow path being distinct from the firstand second horizontal flow paths, a fourth flow path for the sampleprovided at the sample-receiving location, the fourth horizontal flowpath being distinct from the first, second, and third horizontal flowpaths, a first test site with a plurality of distinct and longitudinallydisplaced test lines with one of immobilized antigens or antibodiesassociated with distinct pathogenically unrelated febrile illnesses,preferably all located in a first test zone at a junction of the firstand third sorbent materials, and a second test site with a plurality ofdistinct and longitudinally displaced test lines with one of immobilizedantigens or antibodies associated with the distinct pathogenicallyunrelated febrile illnesses. In one embodiment, the first and secondtest sites includes immobilized antibodies or antigens that distinguishfor different stages of the respective febrile illnesses, e.g., earlyand late stage detection. In another embodiment, the first test siteinclude immobilized antibodies associated with the unrelated febrileillnesses, whereas the second test site include immobilized antigensassociated with the unrelated febrile illnesses. The second test site islocated in a second test zone at a junction of the second and fourthsorbent materials. For purposes herein, the term “distinct” when used inconjunction with the words “flow path” or “migration path” shall beunderstood to mean “not in fluid communication except either (i) via atest zone, or (ii) at a buffer receiving or sample receiving location”.

Where the test cell of the invention is provided in a housing, thehousing is provided with a first opening adjacent the firstbuffer-receiving location and a sample-receiving opening adjacent thesample receiving location. Where a second buffer-receiving location isutilized, a second buffer-receiving opening is provided in the housingadjacent the second buffer-receiving location. A first viewing window isprovided in the housing above the first test line and a second viewingwindow is provided in the housing above the second test line.

In use, a reactive sample, and positive test, may include one of thefirst and second test windows indicating a positive reaction for onlyone or both of the stages of illness for one or more of the plurality offebrile illnesses. In the absence of the antigens or antibodiesindicative of a stage of illness for the febrile illnesses of interest,no associated colored line would be observed under the test window area,and the test would be indicated as negative. The sample continues tomigrate along the second and third sorbent materials and produces acolored line in each of the respective control areas of the first andsecond test windows to indicate that the sample and reagents have beenproperly applied and have migrated through the along the first, secondand third second sorbent materials.

Other multi-assay test designs, provided with the requisite combinationof antibodies or antigens, specific to antigens and/or antibodiesassociated with the plurality of febrile illnesses for which the test isdesigned can be used as well, including wet tests and other constructs.

The assay test is adapted to simply, quickly and accurately determinewhether the carrier of a bodily fluid sample is the subject of one ofplurality of a febrile illnesses. This allows important point-of-caredecisions to be made before additional individual, regional, or even andglobal complications occur.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of a first embodiment of an assay test for testingmultiple unrelated febrile illnesses.

FIG. 1A is a section view across line 1A-1A in FIG. 1.

FIG. 1B is a section view across line 1B-1B in FIG. 1.

FIGS. 2A-2E illustrate use of a febrile illness test.

FIGS. 3A-3E described another embodiment of a febrile illness test,adapted to test for different stages of multiple febrile illnesses.

FIG. 4 is a top view of another embodiment of a febrile illness test.

FIG. 5 is a section view through line 5-5 of FIG. 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to FIGS. 1, 1A and 1B, a dual path lateral flow immunoassaydevice test cell 10 is provided and includes: a housing 20 having a topwall 21 defining first and second holes 24, 26, and a clear plasticwindow 28; and a carrier in the form of first and second sorbent orbibulous materials 30, 32 defining perpendicular horizontal flow pathsin the housing. The first sorbent material 30 includes at least two andpreferably three or four zones and may be made from a plurality ofmaterials. A first zone 31 (sometimes called a filter zone) is locatedat the first hole 24 and extends to a second zone 33 (sometimes called atest zone) which is located at the junction of a “T”. The first zone 31may include a filter 31 a, a pad 31 b on or in which a plurality ofconjugates having desired antigens or antibodies with attached coloredmarkers are deposited and immobilized, and a first portion of a thinmembrane or sorbent or bibulous material 30 typically made fromnitrocellulose with a plastic backing (not shown). The first zone 31 isadapted to receive a buffer solution, to cause the buffer solution tocontact the conjugate, thereby mobilizing the conjugate, and to wick theconjugate-carrying buffer solution to the second zone 33. The second(test) zone 33 includes a second portion of the thin membrane 30 whichis preferably printed with a plurality of longitudinally displaced testlines 50A, 50B, 50C, 50D, 50E (collectively 50A-E) having immobilizedantigens or antibodies (depending on whether the test cell is designedto test for the presence of antibodies or antigens) for unrelatedfebrile illnesses on the membrane. Indicia 70 are provided on the topwall 21 to associate the test lines with a key 72 identifying therespective febrile illnesses under test. The selection of the febrileillnesses for the associated immobilized antigens or antibodiespositioned on respective test lines, is discussed in detail below. Thetest lines 50A-E may be seen through the window 28 provided in thehousing. An optional third zone 35 (sometimes called a control zone)which includes a third portion of the thin membrane 30 may also beprinted with a control line 60 typically containing antibodies to theconjugate antigens (or in some cases antibodies which will bind toconjugate antibodies, or even antigens which will bind to conjugateantibodies) as is well known in the art. Where the third zone 35 isprovided, the window 28 extends above the control line 60; alternativelya separate window may be provided for the control line. If desired, anoptional fourth zone 37 (sometimes called a reservoir zone) may beprovided as a wicking reservoir as is also well known in the art. Thefourth zone 37 includes a relatively thicker absorbent paper 31 d. In anembodiment, overlying all the zones is a thin, transparent plastic filmor card 38 a having an adhesive which keeps the sorbent materials inplace. The card 38 may be cut with an opening at hole 24 so that it doesnot block liquid access to the hole 24.

The second sorbent material 32 may also be made from a plurality ofmaterials and preferably includes two zones 61, 63. The first zone 61(sometimes called a filter zone) includes a filter or pad 62 and a firstportion of a thin membrane or sorbent or bibulous material 32 typicallymade from nitrocellulose with a backing (not shown). The first zone 61is located at the second hole 26 and extends to the second zone 63. Thesecond zone 63 includes a second portion of the thin membrane 32 whichis in contact with the second zone 33 of the first sorbent material 30.As is seen in FIGS. 1A and 1B, the first sorbent material 30 overliesthe second sorbent material 32 such that the membranes are in contactwith each other (as opposed to the backings contacting the membranes oreach other), and such that the test lines 50A-E are effectively locatedbetween the membranes, although in one embodiment the second sorbentmaterial touches the first sorbent material at or adjacent the testlines, but does not overlie the test zones. Thus, test lines 50A-E couldbe printed on the second zone 63 of the second sorbent material 32instead of, or in addition to the second zone 33 of the first sorbentmaterial 30. If desired, a thin plastic film or card 38 b having anadhesive which keeps the second sorbent material in place may beutilized. The sorbent materials may be in the form of strips and may beconstructed from a standard-type nitrocellulose, and provided withdifferent pore sizes to aid in the flow and migration of sample andsolution, as described hereafter. Additional details and variations onthe construction of the test cell, including materials, orientation andconfiguration of the sorbent material, shapes of the test cell housing,etc. are described in U.S. Pat. Nos. 7,189,522, 7569,397, and 7,879,597,all to Esfandiari, which are incorporated by reference herein in theirentireties.

The immunoassay of FIGS. 1, 1A and 1B may be utilized as follows. First,a sample (not shown) possibly containing antibodies (or antigens) isprovided to the second opening or hole 26 and allowed to migrate throughthe second sorbent material 32 to its second zone 63 which is in contactwith the second zone 33 of the first sorbent material 30. The sample ispreferably fingerstick whole blood. However, venous whole blood, serum,and/or plasma can similarly be used.

Optionally, after providing the sample into hole 26, a preferablymeasured amount of liquid such as a buffer solution may be added to hole26 to help in the migration of the sample. Alternatively, the sample andbuffer solution are premixed before being added into hole 26.Regardless, the sample reaches the test lines 50A-E which are printedatop the second zone 33 of the first sorbent material or infusedtherein. After a desired amount of time, by which time the antibodies(or antigens) in the sample (if present) will have had an opportunity tobind to the antigens (or antibodies) immobilized at the test lines50A-E, a preferably measured amount of liquid such as a buffer solution(not shown) is added to the first opening 24. After another period oftime, sufficient to permit the conjugate to migrate to the test lines50A-E (and control site 60 if provided), the test lines 50A-E (andcontrol site 60 if provided) are inspected via window 28 in order todetermine whether the sample is “positive” or not for any of the febrileillnesses for which the test is adapted. Typically, a “positive” testindicating the presence of the antibody (or antigen) in the sample isobtained when both a test line of test lines 50A-E and the control site60 show lines of color. A “positive” test will be indicative for onlythe febrile illness(es) associated with the colored test line(s). A“negative” test indicating the lack of the presence of the antibody (orantigen) in the sample is obtained when only the control site 60 shows aline of color; i.e., none of the test lines 50A-E show color.

Those skilled in the art will appreciate that the immunoassay 10functions as follows. Because the test lines 50A-E are provided withantigens (or antibodies) for febrile illnesses immobilized on amembrane, if the test sample contains antibodies to the antigens (orantigens to the antibodies) for the respective febrile illnesses, theantibodies (or antigens) will bind themselves to the antigens (orantibodies) at the respective test line. Thereafter, when the conjugate39 containing an antigen for the antibody (or antibody for the antigen)coupled to a colored marker is caused to migrate to the test lines50A-E, if the test sample contains the antibodies (or antigens) whichare now held at the test lines 50A-E, the antigen (or antibody) of theconjugate will bind itself to the antibodies (or antigens) and thecolored marker will cause a colored line to appear at the respectivelines 50A-E. If the test sample does not contain antibodies (orantigens), the conjugate will not have the antibodies (antigens) to bindto at the test lines 50A-E, and no colored line will appear at the testsite. On the other hand, because the control line 60 is provided withantibodies (or antigens), the antigens (or antibodies) of the conjugatewill always bind to the antibodies (or antigens) in the control line 60,thereby causing a colored line to appear at the control site 60 if theconjugate reaches the control site 60. Thus, if sufficient buffersolution is provided to the test cell, a colored line should alwaysappear at the control site 60, thereby providing a control for the test.

Now, in accord with one aspect of the invention, the test lines 50A-Eare adapted to indicate presence of one or more febrile illness selectedfrom a plurality of unrelated febrile illnesses. The febrile illnessesindicated by test lines 50A-50E do not share any particularly commonpathogen or other cause for their respective illness states, such that apositive detection of one illness is in no way presumptive, indicativeor in any way etiologically related to a positive outcome for another ofthe febrile illnesses on the test.

In accord with an assay test described herein, the assay test is adaptedto provide presumptive (i.e., confirmatory) detection of at least twounrelated febrile illnesses. In one embodiment, the assay test morepreferably is adapted to provide presumptive detection of at least threeunrelated febrile illnesses. The febrile illnesses can be selected fortesting on the assay using one or more common features of (oralternatively dissimilar features relative to) the one or more of theunrelated febrile illnesses. By way of example, the selection of febrileillnesses for which the test is designed may include a illnesses causedby one or more of the following: distinct viruses, bacterium and/orparasites; infection from distinct viruses, bacterium, parasites orother contagions that are animal borne, whether selected to be animalborne by the same animal (mosquitos) or by different animals (e.g., bat,rodent, mosquito, flea); infection from distinct viruses, bacterium,parasites, or other contagions that can be aerosolized for transmission;infection from viruses, bacterium, parasites or other contagions thatare transmitted from direct contact; infection from viruses, bacterium,parasites or other contagions that are generally transmitted in thetropics and/or subtropics; infection from a virus, bacteria, parasitesharing one or more related features and which causes a febrile illness.The selection of febrile illness in a test may optionally include a setor subset of illnesses meeting any one or more criteria.

It is specifically recognized that the febrile illnesses tested by theassay test do not share any particularly common cause for theirrespective disease states. That is, there is no known pathogenicpattern, pathogenic causation, or other pathogenic relationship from oneillness to the other. As such, to the extent the test provides apositive detection for one febrile illness, it is expected that only onepositive detection for a febrile illness will be indicated on any onetest. It is, of course, possible that a particular test sample, whentested, can provide positive detection of two or even more febrileillnesses. But given the lack of relationship of the febrile illnessesfor which the test is administered, such results would not necessarilybe expected or a part of the design of the test. Nonetheless, it isanticipated that the unrelated febrile illnesses which can be detectedon any one test will nonetheless have a rationale in the arrangement bywhich they are together tested; i.e., to facilitate and expedite earlydetection and diagnosis of febrile illness at point of detection orpoint of care facility.

In accord with that aspect of the assay test, the febrile illnessesdetected by the assay test may be linked by those febrile illnesseshaving a prevalence within a geographical proximity (by of example,within a state, or within a country, or within neighboring countries, orwithin proximate countries, or within a continent, or within a definedrange of latitude, such as ±20° latitude about the equator, or asbounded by geological formations including rivers and/or mountainsand/or valleys, or an geographical area subject to a common or likeenvironmental conditions or a disaster, or geopolitical hardships, orother geographically definable boundary), and/or those febrile illnesseshaving a prevalence within a population in a given time period, and/orthose febrile illnesses to which a group or population may be subject(by way of example, general population of western Africa, or aid workersof one or more relief organizations, whether localized or scattered, orinhabitants of one or more temporary or semi-permanent shelter orhousing systems, including shelter or housing established by the UnitedNations or another relief aid organization), and/or those febrileillnesses which are potentially borne by travelers at a point of entryat a geographical location (e.g., air travelers at an airport or seatravelers at a sea port), and/or those febrile illnesses that may bepotentially the subject to of a bioterror attack (e.g., can betransmitted in an aerosolized manner). For each of the potential groupsidentified above, an assay test may be designed to test for febrileillnesses that are appropriate for the circumstance. In accord with theinvention, the febrile illnesses are unrelated illnesses, each having adifferent causative factor. In accord with the invention, it is possiblethat the unrelated febrile illnesses selected for a test may have anon-causal logical relationship.

By way of example, as shown in the key 72 on the assay test 10 of FIG.1, the assay test can be designed to test for the Ebola Virus (which isa viral infection and can be animal borne), Plasmodium parasites(Malaria)(which is a parasitic infection and is animal borne bymosquito), Dengue virus (which is a viral infection and is animal borneby mosquito, as well as most common in tropical/subtropical geographicalregions), Yersinia pestis (Plague)(which is a bacterial infection, isanimal borne by rodent and their fleas, is specific to identifiedendemic regions, and can be in aerosol form), Burkholderia pseudomallei(Melioidosis)(which is a bacterial infection, a tropical illness, andcan be in aerosol form or in contaminated soil). These febrile illnesseshave no etiological relationship. A subset thereof are commonlybacterial infections: Pague and Medioidosis, though caused by differentbacteria. A subset thereof are tropical: Dengue virus and Melioidosis,through caused by different agents. A subset thereof are animal borne:Ebola, Malaria, Dengue, and Plague, through borne by different animals(mosquito, rodent and fleas). A subset are distinguished as transmittedby different means: virus (Ebola, Dengue), bacteria (Plague,Melioidosis), and parasite (Malaria). From the foregoing, it appreciatedthat the febrile illnesses under test are unrelated. It is appreciatedthat another combination of unrelated febrile illnesses may be testedtogether, including a subset of the illnesses discussed above togetherwith one or more of, by way of example, Anthrax, Lassa fever, Tularemia,Leptospirosis, Marburg hemorrhagic Fever, Rickettsial disease, Thyphoid,Chikungunya, Coxiella burnetii bacteria (Q-fever), Meningococcal,Pneomococcus, and Crimean-Congo haemorrhagic fever (CCHF).

In accord with embodiments of the assay, and not by way of limitation,the assay is adapted to detect for at least three of, or all of, EbolaVirus, Plasmodium parasites (Malaria), Dengue virus, as well as thebacteria Yersinia pestis (Plague) and Burkholderia pseudomallei(Melioidosis) in a bodily fluid. In accord with the preferred assaytest, the assay is preferably adapted to use the bodily fluid offingerstick whole blood, venous whole blood, serum and/or plasma.

Turning now to FIGS. 2A through 2E, another test cell 110 substantiallysimilar to the test cell 10 is shown with like parts having relatedreference numerals. Test cell 10′ includes a housing including a topshell 21′. The sorbent strips are preferably substantially arranged asdescribed above in FIG. 1. The top shell 21′ defines a first hole 24′ inthe form of a well for receiving a buffer (B) solution onto the firstsorbent strip 30′, and a second hole 26′ in the form of a well forreceiving a mixture of the sample and buffer solution (S+B) onto thesecond sorbent strip 32′. The first sorbent strip 30′ is marked withfive test lines (marked “1 2 3 4 5” on the shell 21′), each test linecorresponding to one of the antigens (or antibodies) for one of theunrelated febrile illnesses under test. Strip 30′ also includes anotherline functioning as a control line (marked C). The test lines areprovided with a color dye (e.g., blue) so that they are visible. Thecontrol line is provided with a color dye (e.g., green) so that it isvisible. A first window 35 a′ is provided in the housing over the testlines and a second window 35 b′ is provided in the housing over thecontrol line; however such window may be combined in a communal opening.

In operation, fingerstick whole blood or other suitable bodily fluidsample is obtained and mixed with buffer solution. Then, referring toFIG. 2B, approximately 100 of blood and buffer mixture is added to thesame sample well (marked S+B). Migration of the sample (typically withthe help of the buffer) to the test zone will cause the dye at all thetest lines and as well as the control line to dissipate. Disappearanceof the dye from the lines confirms that the sample has reached the testarea. Once the lines have completely disappeared, as shown at FIG. 2B,approximately 120 μl of buffer is added to the buffer opening (marked B)in order to cause migration of a conjugate marker to the test area.After a period of time, typically 20 minutes, results may be read. InFIG. 2C, only the control line C is seen, thereby indicating a validresult and that the sample tested negatively for each of the febrileillnesses for which the sample was tested; in this case Ebola, Malaria,Dengue, Plague, and Melioidosis. In FIG. 2D, the control line and line 5are seen, thereby indicating a valid result that the sample testedpositively for Melioidosis antibodies, but negatively for the antibodiesfor Ebola, Malaria, Dengue, and Plague. In FIG. 2E, the control line andlines 1 and 3 are seen, thereby indicating a presumed unusual butnonetheless valid result: the sample tested positive for Ebola andDengue antibodies, but negatively for the antibodies for Malaria,Plague, and Melioidosis. It is noted that if the control line C is notseen, the test results are not interpreted as being valid.

Turning now to FIGS. 3A-3D, another embodiment of a multiple unrelatedfebrile illness test 110 is shown. The test cell includes a first teststrip 130 a having a fluid pathway along which a test site 150 aprinted. The test site 150 a has multiple test lines (1 2 3 4 5) and acontrol line (C). The first strip 130 a also has a liquid bufferreceiving portion 124 a, and immobilized conjugates 139 a that arereleased to migrate along the fluid pathway and toward the test sitewhen the liquid receiving portion receives a liquid solution, all suchfeatures substantially as described as with respect to sorbent material30 in the embodiment described in FIGS. 1, 1A and 1B. The test lines ofthe test site 150 a on the first test strip 130 a are adapted to testfor antibodies (or antigens) for an EARLY (first) stage of a pluralityof febrile illnesses. As such, and by way of example, in an embodiment atest line is provided that detects IgM antibodies to the antigen ofEbola, which can be used to confirm early stages of Ebola; i.e., withina few days after the symptoms begin.

The test 110 also includes a second test strip 130 b, substantiallysimilar to and displaced from the first test strip, and having a secondtest site 150 b. The test lines at the second test site 150 b areadapted to test for antibodies (or antigens) for a LATE (second) stageof the plurality of febrile illnesses. By way of example, in anembodiment, the LATE stage test strip is adapted to detect IgGantibodies to the antigen of Ebola which can be used to confirm a latercourse of the disease, or even indicate that recovery is occurring. Thedetection of IgG and IgM antibodies to the antigens of Ebola isdescribed in U.S. Pat. No. 7,189,522, previously incorporated byreference herein.

A third sorbent strip 170 extends between the first and second sorbentstrips 130 a, 130 b, and contacts the first and second strips at therespective test sites 150 a, 150 b of the first and second strips. Thethird sorbent strip 170 includes a zone 172 in which to receive a commonsample and buffer (S+B) that feeds to the test sites 150 a, 150 b of thefirst and second test strips 130 a, 130 b. It is appreciated that thefirst, second and third strips can be distinctly formed from each other,or alternatively may be constructed as unitary member.

In use, fingerstick whole blood or other suitable bodily fluid sample isobtained and mixed with buffer solution. Then, referring to FIG. 3A,preferably approximately 100 μl of blood and buffer mixture is added tothe same sample well zone 172 (marked S+B) marked between the arrows.The sample, with the help of the buffer, migrates in opposite directionsaway from sample well toward the first and second test sites 150 a, 150b. Referring to FIG. 3B, once the lines have completely disappeared,preferably approximately 120 μl of buffer is added to each of the bufferliquid receiving area 124 a, 124 b in order to cause migration of theconjugate markers on the first and second strips toward the respectivetest sites 150 a, 150 b. After a period of time, typically 20 minutes,results may be read. In FIG. 3C, only the control line C is seen in eachof the first and second test strips 130 a, 130 b, thereby indicating avalid result and that the sample tested negatively for each stage ofeach of the febrile illnesses for which the sample was tested; in thiscase Ebola, Malaria, Dengue, Plague, and Melioidosis. In FIG. 3D, alongthe EARLY stage detection first test strip, the control line and line 1are seen, thereby indicating a valid result that the sample testedpositively for early stage of Ebola; along the LATE stage detectionsecond test strip, the control line and no test line are seen, therebyindicating a valid result and no later stage illness state detectablefor any of the illnesses. In FIG. 3E, along the EARLY detection teststrip 130 a, the control line and no test lines are seen, therebyindicating a valid result without detection of the early stage of any ofthe illnesses. In FIG. 3E, along the LATE stage detection test strip 130b, the control line and line 1 are seen, thereby indicating a validresult and detection of late stage, and possibly a recovery stage, ofEbola. The test is negative for the antibodies for Malaria, Dengue,Plague, and Melioidosis.

Turning now to FIGS. 4 and 5, a lateral flow immunoassay device testcell 210 is provided for the presumptive detection of infection with oneor more selected unrelated febrile illnesses, to either verify or rejectthe respective illnesses individually. The test cell 210 has a housing220 having a top wall 221 defining a first hole 224, a sorbent orbibulous material 230 defining a horizontal flow path in the housing.The sorbent material 230 includes a plurality of zones preferably madeof a plurality of materials. In one zone, conjugates 239 having desiredantigens or antibodies with attached colored markers are deposited andimmobilized. The antigens are respectively associated with severalindependent and unrelated febrile illnesses, as previously described.The first zone 231 is adapted to receive a sample and buffer solutionthrough the first hole 224, to cause the sample and buffer solution tocontact the conjugate 239 thereby mobilizing the conjugate, and to wickthe conjugate-carrying sample and buffer solution to the second zone233. The second (test) zone 233 is preferably printed with a pluralityof longitudinally displaced test lines 250A-E at which are immobilizedantigens or antibodies (depending on whether the test cell is designedto test for the presence of antibodies or antigens) as is well known inthe art. A window 228 of clear plastic is preferably provided over thetest lines 250A-E. Indicia 270, 272 are provide to associate the testline 250A-E with the respective illnesses under test. Additional zonesmay be provided to the test to aid in movement, binding, wicking,solution/fluid reception, solution/fluid storage, or viewing of the testlines. An optional third zone 35 (sometimes called a control zone) mayalso be printed with a control line 260 typically containing antibodiesto the conjugate antigens (or in some cases antibodies which will bindto conjugate antibodies, or even antigens which will bind to conjugateantibodies) as is well known in the art. Where the third zone 235 isprovided, the window 228 of preferably extends above the control line260. If desired, an optional fourth zone 237 (sometimes called areservoir zone) may be provided as a wicking reservoir as is also wellknown in the art. The fourth zone 237 includes a relatively thickerabsorbent paper 241d.

As yet another alternative, test lines for both early and later stageforms of a febrile illness can be co-located on a common test strip orsorbent material, such that each of the early and later stage forms canbe presumptively confirmed on a single test strip. By way of example,early and later stage forms of three unrelated febrile illness can betested with a test zone having six test lines, and early and later stageforms of five unrelated febrile illness can be tested with a test zonehaving ten test lines on a single strip.

The antigens are respectively associated with unrelated,epidemiologically independent febrile illnesses. The febrile illnessesshare a common condition of a high grade fever, and potentiallyadditional considerations. The febrile illnesses are epidemiologicallyindependent as each illness does not share a common biological factor inits cause. However, it may be advisable to test for the febrileillnesses together to confirm one of a potential identity of febrileillnesses, or alternatively collectively rule out all such febrileillnesses, at one time.

There have been described and illustrated herein several embodiments ofimmunoassays and methods of their use. While particular embodiments ofthe invention have been described, it is not intended that the inventionbe limited thereto, as it is intended that the invention be as broad inscope as the art will allow and that the specification be read likewise.Thus, while the specification discusses ligand binding usingantigen/antibody reactions, other ligand binding mechanisms such asaptamer binding, nucleic acid binding, enzymatic binding, etc. may alsobe used. Also, while the test cells are described as having variousnumbers of lines for testing for a corresponding number of ligands, itwill be appreciated that different numbers of lines may be utilized fortesting for different numbers of ligands. In such a case, a singlehousing may be utilized with a single hole for the sample, oralternatively, multiple holes could be utilized if desired. Wheremultiple holes are utilized, multiple strips may be used for one or moresamples provided. Preferably, the multiple strips would touch (e.g.,overlie or underlie) a single strip providing a migration path for theconjugate. It may also be possible to provide a single hole which sitsover or leads to two adjacent strips adapted for sample migration.Further, while the test cells are described as having holes in the topwall of a housing for receiving the sample and the buffer-solution orbuffer-conjugate subsystem, it will be appreciated that one or bothholes may be provided in the end wall or side wall of the housing.Moreover, while it is preferred that the test utilized a lateral flowformat with a bibulous material as a carrier, it is appreciated thatother test constructs, including those using a different solid statecarrier or even a liquid carrier, can be utilized to perform the testsdescribed herein as well.

Also, while the test cell is described is being utilized in conjunctionwith a sample of fingerstick whole blood, it is appreciated that otherbodily fluid can be used as well in the test cell, including venouswhole blood, serum and/or plasma. Additionally, the assay test can beadapted for saliva, vomit, urine, fecal matter, or other bodilydischarge, each of which is considered a bodily fluid for purposesherein. It is appreciated that depending upon the sample an appropriatediluent or other additive may need to be combined with the bodily fluidto allow the bodily fluid to properly migrate on the sorbent materialand otherwise be reactant within the test.

Those skilled in the art will also appreciate that the housing may bemodified in additional ways to include separate windows for each testline. Also, while the invention was described in conjunction with theuse of a buffer solution which is added to the migration path of theconjugate and optionally to the migration path of the sample, it will beappreciated that that one or more buffers may be chosen as desired to beadded to the migration paths depending upon the test or tests to beconducted. Thus, buffers such as phosphate buffers or TRIS (trishydroxymethylaminomethane) buffers are often utilized. However, theinvention is intended to encompass the use of any diluent includingwater. In addition, the diluent may, if needed, may be added to andmixed with the sample prior to adding the sample to the sorbent materialor the sample may be deposited first and the diluent may be addedthereafter. Likewise, any diluent capable of causing conjugate tomigrate may be utilized, and may be premixed with the conjugate in aliquid conjugate system, or provided to the migration path for theconjugate in a dry conjugate system. It will therefore be appreciated bythose skilled in the art that yet other modifications could be made tothe provided invention without deviating from its spirit and scope asclaimed.

What is claimed is:
 1. A test device for the qualitative detection ofone febrile illness from a plurality of pathogenically unrelated febrileillnesses, the test device for use with a solution and for use with aconjugate having a marker, the test device for determining the presenceof one of a plurality of ligands respectively associated with one of theplurality of pathogenically unrelated febrile illnesses in a liquidsample so as to identify which, if any, of the pathogenically unrelatedfebrile illnesses is detected in the test sample, comprising: a) asorbent material having a first width and a first length substantiallylonger than said first width, and a first location for receiving thesolution and defining a first migration path for the solution andconjugate; and b) a test site located on or in the sorbent materialhaving a plurality of separately located immobilized ligand-bindingmechanisms for receiving the liquid sample, at least one ligand-bindingmechanism associated with each of the plurality of ligands for thepathogenically unrelated febrile illnesses.
 2. A test device accordingto claim 1, wherein: at least two of the febrile illnesses are prevalentwithin a geographical proximity.
 3. A test device according to claim 1,wherein: at least one of the febrile illnesses is a viral infection andat least one of the febrile illnesses is a bacterial infection.
 4. Atest device according to claim 1, wherein: each of febrile illnesses iscaused by a different type of bacteria, virus, or parasite.
 5. A testdevice according to claim 1, wherein: at least a plurality of thefebrile illnesses are animal borne by different animals.
 6. A testdevice according to claim 5, wherein: at least one of the febrileillnesses is borne by at least one of a bat, a rodent, a mosquito, and aflea, and at least another of the febrile illnesses is borne by adifferent animal.
 7. A test device according to claim 1, wherein: atleast one of the febrile illnesses can be transmitted by an aerosolizedpathogenic agent.
 8. A test device according to claim 1, wherein: atleast one of the febrile illnesses can be transmitted by direct contact.9. A test device according to claim 1, wherein: at least one of thefebrile illnesses is a tropical and/or subtropical illness.
 10. A testdevice according to claim 1, wherein: each of the febrile illnesses iscaused by a different type of bacteria, virus, or parasite, at least aplurality of the febrile illnesses are animal borne by differentanimals, and at least a plurality of the febrile illnesses are tropicaland/or subtropical illnesses.
 11. A test device according to claim 10,wherein: at least two of the febrile illnesses are prevalent in a commongeographical region.
 12. A test device according to claim 10, wherein:at least one of the febrile illnesses can be transmitted through air andat least one of the febrile illnesses can be transmitted by directcontact.
 13. A test device according to claim 1, wherein: the ligandbinding-mechanism are adapted to bind with at least three of EbolaVirus, Malaria, Dengue virus, Plague, Melioidosis, Anthrax, Lassa fever,Tularemia, Leptospirosis, Rickettsial disease, Thyphoid, Chikungunya,Marburg hemorrhagic Fever Q-fever, Meningococcal, Pneomococcus, andCrimean-Congo haemorrhagic fever (CCHF).
 14. A test device according toclaim 1, wherein: the plurality of ligand binding-mechanism arerespectively adapted to bind with antigens or antibodies for at leastthree of Ebola Virus, Malaria, Dengue virus, Plague, and Melioidosis.15. A test device according to claim 1, wherein: time is required forthe liquid sample to laterally flow from the first location to the testsite such that the liquid sample does not immediately wet the test siteupon application.
 16. A test device according to claim 1, wherein: saidseparately located immobilized ligand-binding mechanisms are related tobioterrorism pathogens.
 17. A test device according to claim 1, furthercomprising: c) a second sorbent material having a second width and asecond length substantially longer than said second width, the secondsorbent material distinct from the first sorbent material and defining asecond migration path distinct from the first migration path, the secondsorbent material intersecting the first sorbent material at or adjacentthe test site, the second sorbent material having a second location forreceiving the liquid sample, wherein time is required for the liquidsample to laterally flow from the second location to the test site suchthat the liquid sample does not immediately wet the test site uponapplication.
 18. A test device for the qualitative detection of onefebrile illness from a plurality of pathogenically unrelated febrileillnesses, the test device for use with a solution and for use with aconjugate having a marker, the test device for determining the presenceof one of a plurality of ligands respectively associated with one of theplurality of febrile illnesses in a liquid sample so as to identifywhich, if any, of the unrelated febrile illnesses is detected in thetest sample, comprising: a) a first sorbent material having a firstwidth and a first length substantially longer than said first width, anda first location for receiving the solution and defining a firstmigration path for the solution and conjugate; b) a test site located onor in the first sorbent material having a plurality of immobilizedligand-binding mechanisms, at least one ligand-binding mechanismassociated with each of the plurality of ligands for the febrileillnesses, wherein the febrile illness are related by having prevalencewithin a geographical proximity; and c) a second sorbent material havinga second width and a second length substantially longer than said secondwidth, the second sorbent material distinct from the first sorbentmaterial and defining a second migration path distinct from the firstmigration path, the second sorbent material intersecting the firstsorbent material at or adjacent the test site, the second sorbentmaterial having a second location for receiving the liquid sample,wherein time is required for the liquid sample to laterally flow fromthe second location to the test site such that the liquid sample doesnot immediately wet the test site upon application.
 19. A test deviceaccording to claim 18, wherein: the febrile illnesses are related byhaving prevalence within a common country.
 20. A test device accordingto claim 17, wherein: the febrile illnesses are related by havingprevalence within neighboring countries.
 21. A test device according toclaim 17, wherein: the febrile illnesses are related by havingprevalence within a continent.
 22. A test device according to claim 17,wherein: the febrile illnesses are related by having prevalence within a±20° of latitude.
 23. A test device according to claim 17, wherein: thefebrile illnesses are related to are related to bioterrorism pathogens.